PROJECT SUMMARY The development of biotherapeutic proteins (i.e., ?biologics?) has been impressive over the past three decades, achieving close to 400 products and an annual market value estimated as much as $500 billion. As a caveat, the biomanufacturing industry has not fully mastered the production of many of these drugs, especially from the perspective of glycosylation, which influences the safety, product stability, productivity, biological activity, downstream processing, pharmacodynamics, and ultimately clinical efficacy of biotherapeutic proteins. For example, limitations of industry-standard Chinese hamster ovary (CHO) cell production systems threaten to slow the growth of biologics, in particular for non-conventional products such as enzymes and ? in the case of this project ? for IgG antibodies with atypical glycosylation. To explain briefly, most (from 75 to 85%) IgG antibodies have a single site of glycosylation that is ?buried? between the two heavy chains in the Fc region of the antibody and thus has a relatively minor impact on the antibody?s pharmacological properties. The remaining 15 to 25% of IgG antibodies have additional ?atypical? glycans in the Fab region, which confound the usual pathways for clinical translation and drug development in industry. As a result, commercialization of this class of antibodies is virtually non-existent, with loss of drug candidates that could potentially treat disorders ranging from rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, to Ebola and cancer. Additional opportunities are lost by not being able to ?build-in? glycans that (almost always) improve pharmacokinetic properties and (occasionally) enhance bioactivity. To overcome these obstacles, this project will design (in Aim 1 of our proposal), develop (Aim 2), and synthesize (Aim 3) these agents using an integrated glycoengineering platform where two antibodies in early development will be glycoengineered by building-in three (or more) N-glycans. Our goals are to not only facilitate the clinical translation of this new class of therapeutic antibodies but also to elucidate design principles that can be mapped broadly onto IgG type antibodies including those with conventional Fc region glycosylation. Our pilot studies have produced our early-stage antibody drug candidates in human embryonic kidney (HEK293F) cells, which provide a higher level and quantitatively different type of glycosylation (i.e., ?2,6- sialylation) than industry-standard Chinese hamster ovary (CHO) cells now used to produce >90% of antibody drugs. To obtain ?humanized? sialylation in CHO cells, we implement a biomanufacturing platform that consists of genetically-modified CHO cells that stably express key glycogenes (e.g., human ?2,6-sialyltransferase) supplemented with a proprietary ?high flux? metabolic precursor for sialic acid biosynthesis that our team has developed over the past several years to control glycosylation to produce clinical grade, glyco-optimized antibodies.